Serine proteases degrade airway mucins in cystic fibrosis

Abstract

Airway mucins are the major molecular constituents of mucus. Mucus forms the first barrier to invading organisms in the airways and is an important defense mechanism of the lung. We confirm that mucin concentrations are significantly decreased in airway secretions of subjects with cystic fibrosis (CF) who have chronic Pseudomonas aeruginosa infection. In sputum from CF subjects without a history of P. aeruginosa, we found no significant difference in the mucin concentration compared to mucus from normal controls. We demonstrate that mucins can be degraded by synthetic human neutrophil elastase (HNE) and P. aeruginosa elastase B (pseudolysin) and that degradation was inhibited by serine proteases inhibitors (diisopropyl fluorophosphates [DFP], phenylmethylsulfonyl fluoride [PMSF], and 1-chloro-3-tosylamido-7-amino-2-heptanone HCl [TLCK]). The mucin concentration in airway secretions from CF subjects is similar to that for normal subjects until there is infection by P. aeruginosa, and after that, the mucin concentration decreases dramatically. This is most likely due to degradation by serine proteases. The loss of this mucin barrier may contribute to chronic airway infection in the CF airway.

Fig. 1.

(A) CF sputum electrophoresis by Western blotting on a 1% agarose gel, blotting to nitrocellulose membranes, and probing with MUC5AC (left) or MUC5B (right) antibody. For patient details, see Table S1 in the supplemental material. The internal standard was used to compare different blots. Group 1, “no colonization”; group 2, “intermittent infection”; group 3, “chronic infection”; group 4, “exacerbation”; and normal ETT control. (B) Mucin concentration. Sputum from 9 subjects with CF who had no positive culture for P. aeruginosa or BCC over the last 8 sequential clinic sputum collections (CF, no colonization), 21 CF subjects who had 1 to 4 positive cultures for P. aeruginosa or BCC over at last 8 sequential clinic sputum collections (CF, intermittent infection), 6 CF subjects who had persistent P. aeruginosa or BCC cultures in sequential clinic sputum collections over at last 8 sputum analyses over at least 2 years (CF, chronic infection), and 16 subjects during an acute pulmonary exacerbation with positive P. aeruginosa or BCC culture (CF, exacerbation), compared with mucus from 11 normal ETT control subjects (control). The results are shown as mean density of individual samples related to the internal control (= 100% relative concentration). *, significant in comparison to “CF, no colonization” (P < 0.05); #, = significant in comparison to “control” (Mann-Whitney U test, P < 0.05).

Fig. 2.

Total DNA concentrations for subjects grouped as described the in text and the legend to Fig. 1B. CF, no colonization, n = 9; CF, intermittent infection, n = 21; CF, chronic infection, n = 6; CF, exacerbation, n = 16; normal controls, n = 11. *, significant in comparison to normal mucus (control); #, significant in comparison to “no colonization” (P < 0.05).

Fig. 3.

Analysis of MUC5AC and MUC5B by Western blotting in sputum incubated at 37°C for 1, 2, 3, 6, and 24 h. Sputum was obtained from normal controls (ETT control), from CF subjects without any history of P. aeruginosa colonization (CF, no colonization), and from CF subjects with chronic P. aeruginosa infection (CF, chronic infection). After 24 h of incubation, mucin was detectable only in control mucus and in sputum from CF subjects without a history of P. aeruginosa colonization.

Fig. 4.

Normal ETT control mucus was incubated for 3 h at 37°C with or without HNE and Pseudomonas elastase B (pseudolysin). The mucin concentration of the native control was set to 100%.

Fig. 5.

Analysis of sputum MUC5AC and MUC5B by Western blotting after incubation at 37°C over 6 h in the presence or absence of proteases inhibitors. Sputum was obtained from a CF subject chronically infected by P. aeruginosa. The mucin concentration of the native control without incubation was set to 100%, and results are presented relative to this. We used the serine protease inhibitors DFP, PMSF, and TLCK, the metalloprotease inhibitors EDTA and GM6001, and the cysteine protease inhibitors leupeptin and E64.